<jats:title>Abstract</jats:title><jats:p>The antifungal and cytotoxic metabolites vioprolides A‐D (<jats:bold>1</jats:bold>–<jats:bold>4</jats:bold>) were obtained from the myxobacterium <jats:italic>Cystobacter violaceus</jats:italic> strain Cb vi35 by fermentation in the presence of the adsorber resin XAD‐1180. Peptolide structures <jats:bold>1–4</jats:bold> composed of eight amino acids and a glyceric acid were determined by spectroscopic methods and total hydrolysis. The absolute configurations of the building blocks were established by GC analysis on chiral stationary phases. Common building blocks of <jats:bold>1–4</jats:bold> are <jats:sc>l</jats:sc>‐Ala, <jats:sc>l</jats:sc>‐Cys, <jats:sc>D</jats:sc>‐Leu, <jats:sc>l</jats:sc>‐Thr, <jats:sc>l</jats:sc>‐MeVal, (<jats:italic>E</jats:italic>)‐dehydrobutyrine (<jats:italic>E</jats:italic>‐Dhb), and <jats:sc>l</jats:sc>‐glyceric acid (<jats:sc>l</jats:sc>‐Gla). Two variable positions are occupied by <jats:sc>l</jats:sc>‐Pro or <jats:sc>l</jats:sc>‐Homopro and <jats:sc>l</jats:sc>‐Pro or <jats:italic>trans</jats:italic>‐(2<jats:italic>S</jats:italic>,4<jats:italic>R</jats:italic>)‐4‐methylazetidinecarboxylic acid [(2<jats:italic>S</jats:italic>,4<jats:italic>R</jats:italic>)‐Maz]. Two defined isomers of <jats:bold>1</jats:bold> were detected in CDCl<jats:sub>3</jats:sub> in a ratio of 3 : 2 (2D‐<jats:sup>1</jats:sup>H NOESY) and assigned to rotational isomers at the amide bond of ThrMeVal.